Inhibitor for adhesion of metal hydroxide-containing substance to waste liquor line of automatic analyzer

ABSTRACT

The present invention provides an inhibitor for adhesion of a metal hydroxide-containing substance to a waste liquid line of an autoanalyzer comprising a polysaccharide; a method of inhibiting adhesion of a metal hydroxide-containing substance to a waste liquid line of an autoanalyzer wherein the inhibitor is used; a reagent for measuring a biological component in a sample, which is effective for inhibiting adhesion of a metal hydroxide-containing substance to a waste liquid line, said reagent containing the said inhibitor; and a method for measuring a biological component in a sample wherein the reagent for measuring a biological component is used.

TECHNICAL FIELD

The present invention relates to an inhibitor for adhesion of a metalhydroxide-containing substance to a waste liquid line of autoanalyzer, amethod of inhibiting adhesion of a metal hydroxide-containing substanceto a waste liquid line of autoanalyzer, a reagent for measuring abiological component in a sample and a method for measuring a biologicalcomponent in a sample.

BACKGROUND ART

In clinical diagnosis, a continuous measurement of many samples using anautoanalyzer has been commonly carried out. When a biological componentin a sample is measured with an autoanalyzer, a reaction cell is washedwith an alkaline washing solution after the reaction of the sample witha reagent for measuring a biological component in the reaction cell. Theresulting reaction solution after the reaction of the sample with thereagent for measuring a biological component and the resulting solutionafter washing the reaction cell with an alkaline washing solution arediscarded through a waste liquid line.

When a metal ion, particularly a divalent metal ion, is contained in thereagent for measuring a biological component, however, a metalhydroxide-containing substance which is insoluble or hardly soluble inwater is produced after washing with an alkaline washing solution, andthe substance is adhered to a waste liquid line. Accordingly, when manysamples are continuously measured using an autoanalyzer, the metalhydroxide-containing substance adhered to the waste liquid line isgradually accumulated and, as a result, the following problems arisesuch as pollution of the reaction cell due to dropping of the metalhydroxide-containing substance adhered to the waste liquid line into thereaction cell, pollution of the reaction cell due to overflow of thealkaline washing solution caused by accumulation of the metalhydroxide-containing substance in the waste liquid line, reduction inmeasurement accuracy and influence on the measurement of other items tobe measured due to pollution of the reaction cell, reduced measuringefficiency resulted from interruption of the continuous measurement dueto clogging of the waste liquid line caused by the metalhydroxide-containing substance, and an excessive load to theautoanalyzer, etc.

DISCLOSURE OF THE INVENTION

An object of the present invention is to provide an inhibitor foradhesion of a metal hydroxide-containing substance to a waste liquidline in a continuous measurement of many samples using an autoanalyzer,a method of inhibiting adhesion of a metal hydroxide-containingsubstance to a waste liquid line of an autoanalyzer, a reagent formeasuring a biological component which is effective for inhibition ofadhesion of a metal hydroxide-containing substance to a waste liquidline and a method for measuring a biological component which iseffective for inhibition of adhesion of a metal hydroxide-containingsubstance to a waste liquid line.

The present invention relates to the following (1) to (10).

-   -   (1) An inhibitor for adhesion of a metal hydroxide-containing        substance to a waste liquid line of an autoanalyzer, which        comprises a polysaccharide.    -   (2) The inhibitor according to (1), wherein the polysaccharide        is alginic acid or a salt thereof or dextran.    -   (3) A method of inhibiting adhesion of a metal        hydroxide-containing substance to a waste liquid line of an        autoanalyzer, which comprises using the inhibitor mentioned        in (1) or (2).    -   (4) A reagent for measuring a biological component in a sample,        which comprises the inhibitor mentioned in (1) or (2).    -   (5) The reagent according to (4), which comprises a divalent        metal ion.    -   (6) The reagent according to (5), wherein the divalent metal is        a divalent ion selected from the group consisting of a magnesium        ion, a calcium ion, a cobalt ion and a manganese ion.    -   (7) The reagent according to any one of (4) to (6), wherein the        biological component is a component which is measured by a        biochemical means.    -   (8) The reagent according to anyone of (4) to (7), wherein the        reagent for measuring a biological component in the sample is a        reagent used for a measuring method using an autoanalyzer.    -   (9) The reagent according to (8), wherein the measuring method        using an autoanalyzer is a continuous measuring method of many        samples.    -   (10) A method for measuring a biological component in a sample,        which comprises using the reagent mentioned in any one of (4) to        (9).

BEST MODE FOR CARRYING OUT THE INVENTION

With regard to a measuring method using an autoanalyzer according to thepresent invention, there is no particular limitation so far as it is amethod which comprises a step of washing a reaction cell with analkaline washing solution, although a continuous measuring method ofmany samples is preferred. An example of the measuring method using anautoanalyzer according to the present invention includes a methodcomprising:

-   -   (1) a step of adding a sample and a reagent for measuring a        biological component to a reaction cell,    -   (2) a step of reacting the sample with the reagent for measuring        a biological component,    -   (3) a step of measuring a detectable substance produced in the        step (2) and    -   (4) a step of washing the reaction cell with an alkaline washing        solution.

The step of washing the reaction cell with an alkaline washing solutionincludes a step of adding an alkaline washing solution to a reactioncell and a step of removing the resulting solution after washing withthe alkaline washing solution (hereinafter, referred to as a reactionsolution-containing alkaline washing solution) through a waste liquidline. Each of the steps is not always a completely independent. Forexample, when the reagent for measuring a biological component is in aform of a kit comprising two reagents (first reagent and secondreagent), the second reagent may be added after the reaction of thesample with the first reagent.

In a continuous measuring method of samples using an autoanalyzer, afterthe reaction of the sample with the reagent for measuring a biologicalcomponent, the reaction solution in a reaction cell is discarded througha waste liquid line. Then, an alkaline washing solution is added to thereaction cell, and, after washing, the reaction solution-containingalkaline washing solution resulting from the washing of the cell isdiscarded through the waste liquid line. The waste liquid line comprisesa nozzle which sucks a reaction solution in the reaction cell and areaction solution-containing alkaline washing solution in the reactioncell, and a waste liquid tube connecting to the nozzle. With regard tothe alkaline washing solution, there is no particular limitation so faras the solution is an alkaline washing solution which washes a reactioncell of an autoanalyzer, and examples thereof include aqueous solutionscontaining alkali such as lithium hydroxide, sodium hydroxide andpotassium hydroxide. Although there is no particular limitation to theconcentration of the alkali in an alkaline washing solution, and theconcentration is, for example, 0.1 to 5.0 mol/L. The alkaline washingsolution may contain a detergent, etc. if necessary. Examples of thedetergent include cationic detergent, anionic detergent, amphotericdetergent and nonionic detergent.

The autoanalyzer used in the continuous measuring method of many samplesmay be an autoanalyzer in which a waste liquid line for discarding thereaction solution after the reaction of the sample with the reagent formeasuring a biological component in the reaction cell and a waste liquidline for discarding the reaction solution-containing alkaline washingsolution are the same or different. Examples of the autoanalyzer inwhich a waste liquid line for discarding the reaction solution after thereaction of the sample with the reagent for measuring a biologicalcomponent in the reaction cell and a waste liquid line used to discardthe reaction solution-containing alkali washing solution are the sameinclude Hitachi automatic analyzer 7250, Hitachi automatic analyzer 7600and Hitachi automatic analyzer 7170. In a continuous measurement of manysamples using an autoanalyzer in which both waste liquid lines are thesame one, a reaction solution and a reaction solution-containingalkaline washing solution alternately pass through the waste liquidline. In other words, after a reaction solution passes through the wasteliquid line, passing of a reaction solution-containing alkaline washingsolution on the waste liquid line where the reaction solution remainsand passing of the reaction solution on the waste liquid line where thereaction solution-containing alkaline washing solution remains arerepeated alternately.

In the present invention, the metal hydroxide-containing substance maybe anything so far as it is a substance containing a metal hydroxidewhich is insoluble or hardly soluble in water and causes adhesion to thewaste liquid line, and the examples include an alkaline earth metalhydroxide such as magnesium hydroxide, calcium hydroxide, cobalthydroxide and manganese hydroxide and a complex of such an alkalineearth metal hydroxide with an organic or inorganic substance in thereagent for measuring a biological component.

Examples of the polysaccharide used as an inhibitor for adhesionaccording to the present invention include cyclodextrin, pullulan,dextran, alginic acid or a salt thereof and dextran sulfate or a saltthereof, and preferable are dextran, alginic acid or a salt thereof.Examples of the dextran include dextrans having a molecular weight of200,000, 500,000 and 2,000,000. Examples of the salt of alginic acid andthe salt of dextran sulfate include lithium salt, sodium salt, potassiumsalt and ammonium salt.

Although the inhibitor for adhesion according to the present inventionper se can be used as a reagent, it can be used by being contained in areagent for measuring a biological component or in an alkaline washingsolution. When the inhibitor for adhesion according to the presentinvention per se is used as a reagent, there is no particular limitationto the amount of the polysaccharide in the reagent so far as it is anamount with which adhesion of the metal hydroxide-containing substanceonto the waste liquid line is suppressed, and the amount is, forexample, 0.5 to 20 g/L or, preferably, 1 to 10 g/L in the reactionsolution.

When the inhibitor for adhesion according to the present invention iscontained in a reagent for measuring a biological component, there is noparticular limitation to the amount of the polysaccharide in the reagentfor measuring a biological component so far as it does not affect themeasurement of the biological component and adhesion of the metalhydroxide-containing substance to the waste liquid line of anautoanalyzer is suppressed, and the amount is, for example, 0.5 to 20g/L or, preferably, 1 to 10 g/L in the reaction solution.

When the inhibitor for adhesion according to the present invention iscontained in an alkaline washing solution, there is no particularlimitation to the amount of the polysaccharide in the alkaline washingsolution so far as adhesion of the metal hydroxide-containing substanceto the waste liquid line of an autoanalyzer is suppressed, and theamount is, for example, 0.5 to 20 g/L or, preferably, 1 to 10 g/L in thereaction solution.

The reagent for measuring a biological component according to thepresent invention contains the inhibitor for adhesion according to thepresent invention and a substance which is necessary to measure thebiological component in the sample. When the reagent for measuring abiological component contains a divalent metal ion, the inhibitor foradhesion according to the present invention is effective for inhibitionof adhesion of a metal hydroxide-containing substance to a waste liquidline. Thus, when the reagent for measuring a biological componentaccording to the present invention is used, adhesion of a metalhydroxide-containing substance formed in the waste liquid line duringthe reaction of the reaction solution with the alkaline washing liquidcontaining the reaction solution to a waste liquid line can be inhibitedeven in a continuous measurement of many samples. Examples of thedivalent metal ion include a magnesium ion, a calcium ion, a cobalt ionand a manganese ion. There is no particular limitation to the amount ofthe divalent metal ion in the reagent for measuring a biologicalcomponent, and it is, for example, 0.5 to 5 g/L in the reactionsolution.

The form for presence and preservation of the reagent for measuring abiological component according to the present invention may be either ina freeze-dried state or in a state of an aqueous solution, and it may beeither in a form of one-reagent system or in a form of a kit comprisingtwo or more reagents.

Examples of the sample which can be measured for the biologicalcomponents therein using the reagent for measuring a biologicalcomponent according to the present invention include total blood,plasma, serum, cerebrospinal fluid, saliva, amniotic fluid, urine, sweatand pancreatic juice. Examples of the biological components which can bemeasured using the reagent for measuring a biological componentaccording to the present invention include components which aredetermined by a biochemical means, components which are measured by animmunological means and components which are measured by a geneticmeans, and a preferable one is components measured by a biochemicalmeans. Examples of the components measured by a biochemical meansinclude a biological component measured using an enzymatic reaction.More specific examples include glucose, 1,5-anhydroglucitol, fucose,urea, uric acid, ammonia, creatinine, total cholesterol, freecholesterol, cholesterol in high-density lipoprotein (HDL-C),cholesterol in low-density lipoprotein (LDL-C), cholesterol invery-low-density lipoprotein (VLDL-C), cholesterol in remnant-likelipoprotein (RLP-C), triglyceride, phospholipid, total protein, albumin,globulin, bilirubin, bile acid, sialic acid, lactic acid, pyruvic acid,free fatty acid, ceruloplasmin, alanine aminotransferase (ALT),aspartate aminotransferase (AST), creatine phosphokinase (CPK),phosphokinase (PK), amylase, lipase, choline esterase, γ-glutamyltranspeptidase, leucine aminopeptidase, L-lactate dehydrogenase (LDH),aldolase, alkaline phosphatase, acid phosphatase,N-acetylglucosaminidase, guanase and monoamine oxidase.

Examples of the components measured using an immunological means includebiological components measured by an antigen-antibody reaction. Morespecific examples include IgG, IgM, IgA, IgE, apoprotein AI, apoproteinAII, apoprotein B, apoprotein E, rheumatoid factor, D-dimer, oxidizedLDL, glycoalbumin, T3, T4, drugs such as an antiepileptic drug,α-fetoprotein (AFP), carcinoembryonic antigen (CEA), CA19-9, CA125,human chorionic gonadotropin (hCG), insulin, C-peptide, estrogen,anti-GAD antibody, pepsinogen, HB antigen, anti-HB antibody, HCVantigen, anti-HCV antibody, HTLV-I antigen, anti-HTLV-I antibody, HIVantibody, tuberculosis antibody, mycoplasma antibody and hemoglobin A1c.Examples of the components measured by a genetic means includebiological components measured by hybridization. More specific examplesinclude DNA, RNA and peptide nucleic acid.

When a biological component is converted into a detectable substance, ora detectable substance is generated from a biological component by thereagent for measuring a biological component according to the presentinvention, the biological component can be measured by measuring thedetectable substance. Examples of the detectable substance include dye,NAD(P)H, fluorescence and luminescence.

As a substance which converts a biological component into dye, as wellas a substance which by itself converts a biological component into dye,a combination of plural substances may be used and the examples includea combination of a substance which converts a biological component intohydrogen peroxide and a substance which converts the said hydrogenperoxide into dye and a combination of a substance which converts abiological component into NAD(P)H and a substance which converts thesaid NAD(P)H into dye.

As a substance which forms dye from a biological component, a substancewhich by itself forms dye may be used, and a combination of pluralsubstances may be used, and the examples include an enzyme substratewhich generates dye by action of an enzyme in the biological component,a combination of a substance which generates hydrogen peroxide from abiological component and a substance which converts the hydrogenperoxide into dye and a combination of a substance which generatesNAD(P)H from the biological component and a substance which converts thesaid NAD(P)H into dye.

As a substance which converts a biological component into hydrogenperoxide, an oxidase for the component is mentioned, and when an oxidasefor the component is not available, a combination of a substance whichconverts the component into a substance in which the correspondingoxidase is present, and a substance comprising the oxidase, etc. may beused. With regard to a combination of a biological component and asubstance which converts the biological component into hydrogenperoxide, the following combinations may be exemplified.

-   -   choline: choline oxidase,    -   cholesterol: cholesterol oxidase,    -   uric acid: uricase,    -   triglyceride: lipoprotein lipase and glycerol oxidase,    -   free fatty acid: acyl CoA synthetase and acyl CoA oxidase,    -   glucose: pyranose oxidase,    -   phospholipid: phospholipase D and choline oxidase,    -   creatine: creatinase and sarcosine oxidase,    -   creatinine: creatininase, creatinase and sarcosine oxidase,    -   lactic acid: lactate oxidase,    -   inorganic phosphorus: purine nucleoside phosphorylase and        xanthine oxidase,    -   o-toluoylcholine: choline esterase and choline oxidase,    -   monoamine (allylamine, etc.): monoamine oxidase.

As a substance which converts hydrogen peroxide into dye, a combinationof a peroxidative substance such as peroxidase and an oxidative coloringtype of chromogen, etc. may be used. Examples of the oxidative coloringtype of chromogen include a leuco type chromogen, an oxidative couplingcoloring type of chromogen, etc.

A leuco type of chromogen is a substance which is converted into dye byitself in the presence of hydrogen peroxide and a peroxidative substancesuch as peroxidase, and the examples include10-N-carboxymethylcarbamoyl-3,7-bis(dimethylamino)-10H-phenothiazine(CCAP), 10-N-methylcarbamoyl-3,7-bis(dimethylamino)-10H-phenothiazine(MCDP),N-(carboxymethylamino-carbonyl)-4,4′-bis(dimethylamino)diphenylaminesodium salt (DA-64), 4,4′-bis(dimethylamino)diphenylamine andbis[3-bis(4-chlorophenyl)methyl-4-dimethylaminophenyl]amine (BCMA).

An oxidative coupling coloring type of chromogen is a substance whichforms dye by oxidative coupling of two compounds in the presence ofhydrogen peroxide and a peroxidative substance such as peroxidase.Examples of the combination of two compounds include a combination of acoupler and an aniline compound, and a combination of a coupler and aphenol compound. Examples of the coupler include 4-aminoantipyrine(4-AA), and 3-methyl-2-benzothiazolinone hydrazine. Examples of theaniline compound include N-ethyl-N-(3-methylphenyl)-N′-succinylethylenediamine (EMSE), N-(3,5-dimethoxyphenyl)-N′-succinylethylenediamine sodium salt (DOSE),N-ethyl-N-(2-hydroxy-3-sulfopropyl)-m-toluidine,N-ethyl-N-sulfopropylaniline,N-ethyl-N-sulfopropyl-3,5-dimethoxyaniline,N-sulfopropyl-3,5-dimethoxyaniline,N-ethyl-N-sulfopropyl-3,5-dimethylaniline,N-ethyl-N-sulfopropyl-m-toluidine,N-ethyl-N-(2-hydroxy-3-sulfopropyl)-m-anisidine,N-ethyl-N-(2-hydroxy-3-sulfopropyl)-aniline,N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline sodium saltdihydrate (TOOS),N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline,N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline sodium salt (HSDA),N-ethyl-N-(2-hydroxy-3-sulfoproyl)-3,5-dimethylaniline,N-sulfopropylaniline, N-ethyl-N-sulfopropylaniline, propyl-m-anidine andN-ethyl-N-(2-hydroxy-3-sulfopropyl)-4-fluoro-3,5-dimethoxyaniline sodiumsalt (F-DAOS). Examples of the phenol compound include phenol and3-hydroxy-2,4,6-triiodobenzoic acid.

As a substance which converts a biological component into an NAD(P)H,dehydrogenase for the biological component may be mentioned, and whendehydrogenase of the biological component is not available, acombination of a substance which converts the biological component intoa substance in which the corresponding dehydrogenase is present and asubstance comprising the dehydrogenase, etc. may be used. As acombination of a biological component and a substance which converts thebiological component into NAD(P)H, the following combinations may beexemplified.

-   -   Glucose: glucokinase, adenosine triphosphate,        glucose-6-phosphate dehydrogenase and NAD(P)⁺,    -   1,5-Anhydroglucitol: hexokinase or glucokinase, adenosine        diphosphate or adenosine triphosphate,        1,5-anhydroglucitol-6-phosphate dehydrogenase and NAD(P)⁺.

As substance which converts NAD(P)H into dye, a reductive coloring typeof chromogen, etc. is mentioned. Examples of the chromogen of areductive coloration type include3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT),2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazoliummonosodium salt (WST-1) and2-(4-iodophenyl)-3-(2,4-dinitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazoliummonosodium salt (WST-3).

As the enzyme substrate which forms dye by the action of an enzyme,mentioned is 4-nitrophenyl β-D-galactosyl-α-maltopentaoside whichproduces 4-nitrophenol by the action of α-amylase, etc.

As a substance which generates hydrogen peroxide from a biologicalcomponent, a substrate of oxidase may be mentioned when the biologicalcomponent is oxidase, and when the biological component is an enzymewhich is other than oxidase, mentioned is a combination of a substratefor enzyme, a substance which converts a substance produced by thereaction of the enzyme with the substrate into a substance in which thecorresponding oxidase is present, and a substance comprising theoxidase. As a combination of the biological component and a substancewhich generates hydrogen peroxide from the biological component, thefollowing combinations may be exemplified.

-   -   Cholinesterase: 2,4-dimethoxybenzoylcholine and choline oxidase,    -   Monoamine oxidase: allylamine.

As a substance which generates NAD(P)H from a biological component, asubstrate for dehydrogenase may be mentioned when the biologicalcomponent is dehydrogenase, and when the biological component is anenzyme other than dehydrogenase, mentioned is a combination of asubstrate for the enzyme, a substance which converts the substanceproduced by the reaction of the enzyme with the substrate into asubstance in which the corresponding dehydrogenase is present and asubstance comprising the dehydrogenase. As a combination of thebiological component and a substance which generates NAD(P)H from thebiological component, the following combination may be exemplified.

-   -   Creatine phosphokinase: creatine phosphate, adenosine        diphosphate, glucose, NAD(P)⁺ and glucose-6-phosphate        dehydrogenase.

When a substance which is converted from a biological component orgenerated from a biological component is NAD(P)⁺, the biologicalcomponent can be determined by measuring the decrease of NAD(P)H. Whenthe decrease of NAD(P)H is measured, the reagent for measuring abiological component according to the present invention contains NAD(P)Hinstead of NAD(P)⁺.

When a substance which is converted from a biological component orgenerated from a biological component is fluorescence, a substance whichis contained in the reagent for measuring a biological componentaccording to the present invention and which converts the biologicalcomponent into fluorescence or generates fluorescence from thebiological component, a substance which by itself converts intofluorescence or a substance which by itself generates fluorescence maybe used, and a combination of plural substances may be used. Examples ofthe substance which converts a biological component into fluorescence orgenerates fluorescence from a biological component include a combinationof a substance which converts the biological component into hydrogenperoxide and a substance which generates fluorescence from the hydrogenperoxide, a combination of a substance which generates hydrogen peroxidefrom a biological component and a substance which generates fluorescencefrom the hydrogen peroxide, etc.

Examples of the substance which converts a biological component intohydrogen peroxide include the above-mentioned substance which converts abiological component into hydrogen peroxide, etc. Examples of thesubstance which generates hydrogen peroxide from a biological componentinclude the above-mentioned substance which generates hydrogen peroxidefrom a biological component etc. Examples of the substance whichgenerates fluorescence from hydrogen peroxide include a combination of aperoxidative substance such as peroxidase and a fluorescent substance,etc. Examples of the fluorescent substance include 4-hydroxyphenylaceticacid, 3-(4-hydroxphenyl)propionic acid, coumarin, etc.

When a substance which is converted from a biological component orgenerated from a biological component is luminescence, as a substancewhich is contained in the reagent for measuring biological componentaccording to the present invention and which converts the biologicalcomponent into luminescence or a substance which generates luminescencefrom the biological component, a substance which by itself converts intoluminescence or a substance which by itself generates luminescence maybe used, and a combination of plural substances may be used. Examples ofthe substance which converts a biological component into luminescence orgenerates luminescence from a biological component include a combinationof a substance which converts the biological component into hydrogenperoxide and a substance which generates luminescence from the hydrogenperoxide, a combination of a substance which generates hydrogen peroxidefrom the biological component and a substance which generatesluminescence from the hydrogen peroxide, etc.

Examples of the substance which converts a biological component intohydrogen peroxide include the above-mentioned substance which converts abiological component into hydrogen peroxide, etc. Examples of thesubstance which generates hydrogen peroxide from a biological componentinclude the above-mentioned substance which generates hydrogen peroxidefrom a biological component, etc. As the substance which generatesluminescence from hydrogen peroxide, a chemiluminescent substance ismentioned. Examples of the chemiluminescent substance include luminol,isoluminol, lucigenin and acridinium ester.

The reagent for measuring a biological component according to thepresent invention may, if necessary, contain buffer, detergent,antiseptic, agent for elimination of affecting substances, etc. Examplesof the buffer include those with pH from 1 to 11 such as lactate buffer,citrate buffer, acetate buffer, succinate buffer, phthalate buffer,phosphate buffer, triethanolamine buffer, diethanolamine buffer, lysinebuffer, barbiturate buffer, tris(hydroxymethyl)aminomethane buffer,imidazole buffer, malate buffer, oxalate buffer, glycine buffer, boratebuffer, carbonate buffer, glycine buffer and Good's buffer. Examples ofGood's buffer include 2-morpholinoethanesulfonic acid (MES),bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane (Bis-Tris),N-(2-acetamido)iminodiacetic acid (ADA),piperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES),N-(2-acetamido)-2-aminoethanesulfonic acid (ACES),3-morpholino-2-hydroxypropanesulfonic acid (MOPSO),N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES),3-morpholinopropanesulfonic acid (MOPS),N-[tris(hydroxymethyl)methyl]-2-aminoethanesulfonic acid (TES),2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES),3-[N,N-bis(2-hydroxyethyl)amino]-2-hydroxypropanesulfonic acid (DIPSO),N-[tris(hydroxymethyl)methyl]-2-hydroxy-3-aminopropanesulfonic acid(TAPSO), piperazine-N,N′-bis(2-hydroxypropanesulfonic acid) (POPSO),3-[4-(2-hydroxyethyl)-1-piperazinyl]-2-hydroxypropanesulfonic acid(HEPPSO), 3-[4-(2-hydroxyethyl)-1-piperazinyl]propanesulfonic acid[(H)EPPS], N-[tris(hydroxymethyl)methyl]-glycine (Tricine),N,N-bis(2-hydroxyethyl)glycine (Bicine),N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS),N-cyclohexyl-2-aminoethanesulfonic acid (CHES),N-cyclohexyl-3-amino-2-hydroxypropanesulfonic acid (CAPSO) andN-cyclohexyl-3-aminopropanesulfonic acid (CAPS).

Examples of the detergent include cationic detergent, anionic detergent,amphoteric detergent and nonionic detergent. Examples of the antisepticinclude sodium azide and antibiotics. As the agent for elimination ofaffecting substances, potassium ferrocyanide, ferrocene compound, etc.are mentioned for eliminating the affection by bilirubin, and ascorbicacid oxidase, etc. are mentioned for eliminating the affection byascorbic acid.

With regard to a method for measuring a biological component in a sampleusing the reagent for measuring a biological component according to thepresent invention, there is no particular limitation so far as it is amethod for measuring a detectable substance produced by the reaction ofthe sample with the reagent for measuring a biological component, andthe examples include absorptiometry, fluorometry and emissionspectrometry. Also, an endpoint method or a rate method may be mentionedas the method for measuring a biological component in a sample using thereagent for measuring a biological component according to the presentinvention.

As an absorptiometry, mentioned is a method where absorption of dye suchas NAD(P)H produced by the reaction of the sample with the reagent formeasuring a biological component is measured by a spectrophotometer. Asa method for production of dye by the reaction of the sample with thereagent for measuring a biological component, mentioned are a methodwhere hydrogen peroxide which is converted from the biological componentin the sample or generated from the biological component in the sampleis converted into dye by the above-mentioned oxidative coloring type ofchromogen in the presence of a peroxidative substance such as peroxidaseand a method where NAD(P)H converted from the biological component orNAD(P)H generated from the biological component in the sample isconverted to dye by the above-mentioned reductive coloring type ofchromogen in the presence of diaphorase and an electron carrier such as1-methoxy-5-methylphenazium methylsulfate.

As a fluorometry, mentioned is a method where fluorescence produced bythe reaction of the sample with the reagent for measuring a biologicalcomponent is measured by a fluorophotometer. As a method for productionof fluorescence by the reaction of the sample with the reagent formeasuring a biological component, mentioned is a method wherefluorescence is generated by the reaction of the above-mentionedfluorescent substance with hydrogen peroxide converted from thebiological component in the sample or hydrogen peroxide generated fromthe biological component in the sample in the presence of peroxidativesubstance such as peroxidase.

As an emission spectrometry, mentioned is a method where luminescenceproduced by the reaction of the sample with the reagent for measuring abiological component is measured by a luminometer. As a method forproduction of luminescence by the reaction of the sample with thereagent for measuring a biological component, mentioned is a methodwhere luminescence is generated by the reaction of hydrogen peroxideconverted from the biological component in the sample or generated fromthe biological component in the sample with the above-mentionedchemiluminescent substance.

Examples of the present invention will be shown below, although thepresent invention is not limited thereto. The reagents and enzymes usedhere are those of the following manufacturers.

PIPES reagent (manufactured by Dojindo Laboratories), MOPS buffer(Dojindo Laboratories), Triton X-100 (manufactured by Sigma), magnesiumsulfate heptahydrate (manufactured by Wako Pure Chemical Industries),TOOS (manufactured by Dojindo Laboratories), 4-AA (manufactured by KantoKagaku), ATP disodium salt (manufactured by Kyowa Hakko Kogyo), sodiumazide (manufactured by Wako Pure Chemical Industries), sodiumdextransulfate (manufactured by Fluka), sodium cholate (manufactured byTokyo Kasei), sodium alginate (manufactured by Fuji Chemical Industry),dextran (molecular weight: 500,000) (manufactured by Pharmacia),glycerol kinase (manufactured by Asahi Kasei), glycerol-3-phosphateoxidase (manufactured by Asahi Kasei), lipoprotein lipase (manufacturedby Toyobo), ascorbate oxidase (manufactured by Asahi Kasei), modifiedlipoprotein lipase (manufactured by Toyobo), peroxidase (manufactured byToyobo) and modified cholesterol oxidase (manufactured by Kyowa HakkoKogyo).

EXAMPLES Example 1 Reagent for Measuring Triglyceride

Reagent for measuring triglyceride was prepared according to thefollowing compositions. (First Reagent) PIPES buffer (pH 7.4)   9 g/LTriton X-100 0.2% Magnesium sulfate heptahydrate   2 g/L TOOS 0.3 g/LATP disodium salt 2.5 g/L Glycerol kinase   1 kU/L Glycerol-3-phosphateoxidase   8 kU/L Peroxidase  10 kU/L Sodium alginate 1.5 g/L (SecondReagent) PIPES buffer (pH 6.8)   9 g/L Triton X-100 0.2% 4-AA 0.5 g/LLipoprotein lipase   3 kU/L Peroxidase  10 kU/L

Example 2 Reagent for Measuring Triglyceride

Reagent for measuring triglyceride was prepared according to thefollowing compositions. (First Reagent) PIPES buffer (pH 7.4)   9 g/LTriton X-100 0.2% Magnesium sulfate heptahydrate   2 g/L TOOS 0.3 g/LATP disodium salt 2.5 g/L Glycerol kinase   1 kU/L Glycerol-3-phosphateoxidase   8 kU/L Peroxidase  10 kU/L Dextran (molecular weight: 500,000)  5 g/L (Second Reagent) PIPES buffer (pH 6.8)   9 g/L Triton X-100 0.2%4-AA 0.5 g/L Lipoprotein lipase   3 kU/L Peroxidase  10 kU/L

Comparative Example 1 Reagent for Measuring Triglyceride

Reagent for measuring triglyceride containing no inhibitor for adhesionof the present invention was prepared according to the followingcompositions. (First Reagent) PIPES buffer (pH 7.4)   9 g/L Triton X-1000.2% Magnesium sulfate heptahydrate   2 g/L TOOS 0.3 g/L ATP disodiumsalt 2.5 g/L Glycerol kinase   1 kU/L Glycerol-3-phosphate oxidase   8kU/L Peroxidase  10 kU/L (Second Reagent) PIPES buffer (pH 6.8)   9 g/LTriton X-100 0.2% 4-AA 0.5 g/L Lipoprotein lipase   3 kU/L Peroxidase 10 kU/L

Test Example 1 Continuous Measurement of Triglyceride in a Sample withan Autoanalyzer (Hitachi 7250)

A continuous measurement of triglyceride in a sample was carried outusing Hitachi automatic analyzer 7250. In the continuous measurement,the reagent of Example 1 was set in the first channel while the reagentof Comparative Example 1 was set in the second channel, and then acontinuous measurement of the sample was conducted.

A series of operations for one sample from the measurement oftriglyceride in the sample to washing of the reaction cell were carriedout as follows. Namely, the sample (5 μL) and the first reagent (240 μL)were added to the reaction cell, and the mixture was heated at 37° C.for 5 minutes, the second reagent (80 μL) was added thereto, and themixture was heated at 37° C. for 5 minutes. The absorbance of theresulting reaction solution was measured under the condition that thephotometric points were 16-34 and main wavelength/subwavelength was 546nm/700 nm. After the determination, the reaction solution in thereaction cell was discarded through a waste liquid line, and then analkaline washing solution (Hitachi High Alkali) containing sodiumhydroxide as a main ingredient was added to wash the reaction cell, andthe reaction solution-containing alkaline washing solution was discardedthrough the same waste liquid line as above. A series of theseoperations were repeatedly carried out for each sample in a continuousmanner. Next, the condition of the waste liquid line used for thecontinuous measurement when the reagent of Example 1 in the channel 1was used was compared with that when the reagent of Comparative Example1 of the channel 2 was used. The result showed that a continuousmeasurement of about 12,000 samples clogged the waste liquid line anddisrupted the continuous measurement when the reagent of ComparativeExample 1 was used, while a continuous measurement of even 20,000 ormore samples did not clog the waste liquid line when the reagent ofExample 1 was used.

Test Example 2 Continuous Measurement of Triglyceride in a Sample withan Autoanalyzer (Hitachi 7250)

A continuous measurement of triglyceride in a sample was carried out bythe same operations as those described in Test Example 1, except thatthe reagent of Example 2 was used instead of the reagent of Example 1.The result showed that a continuous measurement of about 12,000 samplesclogged the waste liquid line and disrupted the continuous measurementwhen the reagent of Comparative Example 1 was used, while a continuousmeasurement of even 20,000 or more samples did not clog the waste liquidline when the reagent of Example 2 was used.

Example 3 Reagent for Measuring Cholesterol in HDL (HDL-C)

Reagent for measuring cholesterol in HDL (HDL-C) was prepared accordingto the following compositions. (First Reagent) MOPS buffer (pH 7.0)   4g/L Sodium dextransulfate 0.5 g/L Magnesium sulfate heptahydrate   2 g/LTOOS 0.3 g/L Sodium azide 0.2 g/L Peroxidase   5 kU/L Ascorbate oxidase  1 kU/L Sodium alginate   2 g/L (Second Reagent) MOPS (pH 7.0)   4 g/L4-AA 0.5 g/L Sodium cholate   4 g/L Sodium azide 0.2 g/L Peroxidase  10kU/L Modified lipoprotein lipase   1 kU/L Modified cholesterol oxidase  7 kU/L

Comparative Example 2 Reagent for Measuring Cholesterol in HDL (HDL-C)

Reagent for measuring cholesterol in HDL (HDL-C) containing no inhibitorfor adhesion of the present invention was prepared according to thefollowing compositions. (First Reagent) MOPS buffer (pH 7.0)   4 g/LSodium dextransulfate 0.5 g/L Magnesium sulfate heptahydrate   2 g/LTOOS 0.3 g/L Sodium azide 0.2 g/L Peroxidase   5 kU/L Ascorbate oxidase  1 kU/L (Second Reagent) MOPS (pH 7.0)   4 g/L 4-AA 0.5 g/L Sodiumcholate   4 g/L Sodium azide 0.2 g/L Peroxidase  10 kU/L Modifiedlipoprotein lipase   1 kU/L Modified cholesterol oxidase   7 kU/L

Test Example 3 Continuous Measurement of HDL-C in a Sample with anAutoanalyzer (Hitachi 7250)

A continuous measurement of HDL-C in a sample was carried out by thesame operations as those described in Test Example 1, except that thereagent of Example 3 was used instead of the reagent of Example 1 andthat the reagent of Comparative Example 2 was used instead of thereagent of Comparative Example 1. The result showed that a continuousmeasurement of about 5,000 samples clogged the waste liquid line anddisrupted the continuous measurement when the reagent of ComparativeExample 2 was used, while a continuous measurement of even 20,000 ormore samples did not clog the waste liquid line when the reagent ofExample 3 was used.

Industrial Applicability

In accordance with the present invention, adhesion of a metalhydroxide-containing substance to a waste liquid line in a measurementusing an autoanalyzer can be inhibited. Accordingly, pollution of thereaction cell and interruption of the continuous measurement do notoccur. As a result, reduction in measurement accuracy and influence onthe measurement of other measuring items are suppressed, measuringefficiency is improved and an excessive load to an autoanalyzer isreduced.

1. An inhibitor for adhesion of a metal hydroxide-containing substanceto a waste liquid line of an autoanalyzer which comprises apolysaccharide:
 2. The inhibitor according to claim 1, wherein thepolysaccharide is alginic acid or a salt thereof or dextran.
 3. A methodof inhibiting adhesion of a metal hydroxide-containing substance to awaste liquid line of an autoanalyzer, which comprises using theinhibitor according to claim
 1. 4. A reagent for measuring a biologicalcomponent in a sample, which comprises the inhibitor according toclaim
 1. 5. The reagent according to claim 4, which comprises a divalentmetal ion.
 6. The reagent according to claim 5, wherein the divalentmetal is a divalent ion selected from the group consisting of amagnesium ion, a calcium ion, a cobalt ion and a manganese ion.
 7. Thereagent according to claim 4, wherein the biological component is acomponent which is measured by a biochemical means.
 8. The reagentaccording to claim 4, wherein the reagent for measuring a biologicalcomponent in the sample is a reagent used for a measuring method usingan autoanalyzer.
 9. The reagent according to claim 8, wherein themeasuring method using an autoanalyzer is a continuous measuring methodof many samples.
 10. A method for measuring a biological component in asample, which comprises using the reagent according to claim 4.